Abstract:

The effects of ammonium on cell growth, metabolism, protein production and glycosylation were investigated for Chinese hamster ovary (CHO) cells transfected with the human erythropoietin (EPO) gene and grown in serum-free medium during batch culture. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two dimensional electrophoresis. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular weight of 18 kDa. Addition of NH4Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased range of molecular weight and pI. The FACE N-linked oligosaccharide profile showed that the density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The N-linked glycan sequence determination by FACE indicated that EPO contained a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. The molecular heterogeneity of EPO increased during the course of a batch culture. Analysis of the secreted EPO indicated a time-dependent increase in the molecular weight band width of the peptide consistent with degradation rather than changes in the glycan structure. A high glutamine concentration (16-20 mM) in the culture decreased the apparent degradation of the EPO. Glucosamine induced EPO heterogeneity which was significantly different from ammonia. The pI range was increased and extended from 3.5 to 7.5 in 10 mM glucosamine supplemented culture. The addition of ammonia and glucosamine resulted in a significant increase of the intracellular nucleotide sugar pool. The UDP-GNAc pool of ammonium and glucosamine treated cultures accounted for 60% of total nucleotides compared to 9.2% for the control culture. In conclusion, ammonia in the culture medium affected EPO glycosylation which was observed as a reduction of the tetra-antennary and tetra-sialylated oligosaccharide structures. The added ammonia and glucosamine to CHO cell culture had different effects on EPO heterogeneity although both significantly increased intracellular UDP-sugar pool concentrations. There may not be a direct relationship between intracellular nucleotide sugar pool and glycan structures from this study. (Abstract shortened by UMI.)